Methods for designing specific ion channel blockers

ABSTRACT

The present invention relates to a method of designing an ion channel blocker for an ion channel which includes providing an ion channel having an external vestibule portion and raising an antibody, binding portion, probe, or ligand specific to the external vestibule portion of the ion channel, where the antibody, binding portion, probe, or ligand inhibits ion transport through the ion channel. The present invention further relates to a method of inhibiting ion transport through an ion channel, a method for screening a drug for effectiveness as an ion channel blocker, and an antibody, binding portion, probe, or ligand.

This application is a divisional of U.S. patent application Ser. No.09/273,217, filed Mar. 19, 1999, which is hereby incorporated byreference in its entirety and claims the benefit of U.S. Provisionalpatent application Ser. No. 60/079,268, filed Mar. 25, 1998, which ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Cell membranes must allow passage of various polar molecules, includingions, sugars, amino acids, and nucleotides. Special membrane proteinsare responsible for transferring such molecules across cell membranes.These proteins, referred to as membrane transport proteins, occur inmany forms and in all types of biological membranes. Each protein isspecific in that it transports a particular class of molecules (such asions, sugars, or amino acids) and often only certain molecular speciesof the class. All membrane transport proteins that have been studied indetail have been found to be multipass transmembrane proteins. Byforming a continuous protein pathway across the membrane, these proteinsenable the specific molecules to cross the membrane without coming intodirect contact with the hydrophobic interior of the lipid bilayer of theplasma membrane.

There are two major classes of membrane transport proteins: carrierproteins and channel proteins. Carrier proteins bind the specificmolecule to be transported and undergo a series of conformationalchanges in order to transfer the bound molecule across the membrane.Channel proteins, on the other hand, need not bind the molecule.Instead, they form hydrophilic pores that extend across the lipidbilayer; when these pores are open, they allow specific molecules(usually inorganic ions of appropriate size and charge) to pass throughthem and thereby cross the membrane. Transport through channel proteinsoccurs at a much faster rate than transport mediated by carrierproteins.

Channel proteins which are concerned specifically with inorganic iontransport are referred to as ion channels, and include ion channels forsodium, potassium, calcium, and chloride ions. Ion channels which openin response to a change in the voltage across the membrane are referredto as voltage-sensitive ion channels.

Ion channels serve numerous physiological functions in excitable andnonexcitable cells (Catterall, W. A., Science 242:50-61 (1988); Lester,H. A., Annu. Rev. Physiol. 53:477-496 (1991); Jan. L. Y. et al., Annu.Rev. Physiol. 54:537-555 (1992)). They transmit electrical signals togenerate physiological cell responses. With electrophysiologicalrecording techniques, a variety of ionic currents in many kinds of cellshave been observed (Catterall, W. A., Science 242:50-61 (1988); Lester,H. A., Annu. Rev. Physiol. 53:477-496 (1991); Jan. L. Y. et al., Annu.Rev. Physiol. 54:537-555 (1992)). The importance of these ionic currentshas been demonstrated by pharmacological approaches using eithernaturally existing ion channel toxins or inorganic and organic ionchannel blockers (such as local anesthetics). The essentialphysiological roles of ion channels in normal cellular functions havebeen further strengthened by the link of diseases to defects in ionchannel genes (Catterall, W. A., Science 242:50-61 (1988); Lester, H.A., Annu. Rev. Physiol. 53:477-496 (1991); Jan. L. Y. et al., Annu. Rev.Physiol. 54:537-555 (1992)).

Over the past few years, molecular biological studies have revealed alarge number of ion channel genes that could be responsible for theobserved ionic currents (Pongs, O., Physiol. Rev. 72:S69-S88 (1992);Perney, T. M. et al., Semin. Neurosci. 5:135-145 (1993); Chandy, K. G.et al., In CRC Handbook of Receptors and Channels ed. North, R. A. (BocaRaton, Fla.:CRC), pp. 1-71 (1995); Deal, K. K. et al., Physiol. Rev.76:49-67 (1996)). For example, there are more than 20 genes that havebeen cloned coding for voltage-gated potassium channels (Pongs, O.,Physiol. Rev. 72:S69-S88 (1992); Perney, T. M. et al., Semin. Neurosci.5:135-145 (1993); Chandy, K. G. et al., In CRC Handbook of Receptors andChannels ed. North, R. A. (Boca Raton, Fla.:CRC), pp. 1-71 (1995); Deal,K. K. et al., Physiol. Rev. 76:49-67 (1996)). Just within the Kv1subfamily of the voltage-gated K⁺ channels, there are at least sevenmembers, and most of them (except Kv1.4) generate similardelayed-rectifier K⁺ currents. Moreover, different potassium channelsubunits can co-assemble to form heteromultimeric channels (Isacoff, E.Y. et al., Nature 345:530-534 (1990); Ruppersberg, J. P. et al., Nature345:535:537 (1990);Christie, M. J. et al., Neuron 2:405-411 (1990)).Finally, the native complex of voltage-gated K⁺ channels is alsocomposed of accessory β-units and these β-subunits could convert thedelayed-rectifier currents into rapidly inactivating A-type K⁺ currents(Rettig, J. et al., Nature 369:289-294 (1994)).

Antibodies have previously been used in functional studies of channels.Antipeptide antibodies, made against regions between S5 and S6transmembrane segments of domains I and IV of the sodium channelα-scorpion toxin to sodium channels reconstituted in phospholipidvesicles or synaptosomes (Thomsen et al., Proc. Natl. Acad. Sci. USA86:10161-10165 (1989)). It was not shown whether these antibodies couldblock sodium currents. An antipeptide antibody, by binding to a regionin the intracellular loop between domains III and IV, slows sodiumchannel inactivation (Vassilev et al., Science 241:1658-1661 (1988)).Furthermore, it has been found that antisera from patients withLambert-Eaton Myasthenic Syndrome (an autoimmune disease ofneuromuscular transmission) could inhibit calcium channel currents (Kimet al., Science 239:405-408 (1988)). Antisera from some patients withIsaacs' Syndrome (acquired neuromyotonia) have antibodies againstpotassium channels and could increase neuronal excitability, possiblydue to blocking of potassium currents (Shillito et al., Ann. Neuol.38:714-722 (1995)). One monoclonal antibody that was generated againstmembrane fragments of the eel electroplax attenuates sodium current(Meiri et al., Proc. Natl. Acad. Sci. USA 88:8385-8399 (1986)). Anothermonoclonal antibody that recognizes the dihydropyridine-binding complexin rabbit muscle transverse tubules inhibits calcium current in a mousemuscle cell line (Morton et al., J. Biol Chem. 263:613-616 (1988)).However, in all these cases, the binding sites on the channel proteinswas not clear.

The challenge now is to pin-point the underlying molecular identities(ion channel proteins) responsible for the observed ionic currents innative cells and to define their physiological functions. Althoughgenetic manipulation with targeted deletion of ion channel genes wouldbe helpful, the interpretation of results could be complicated byfunctional redundancy and developmental abnormalities. Some ion channelblockers are available, but they usually affect a group of ion channelsand, thus, lack specificity towards one specific channel protein. Theseblockers were found empirically, either by clinical use or by broadfunctional screening, rather than by rational design. The presentinvention is directed to overcoming these deficiencies.

SUMMARY OF THE INVENTION

The present invention relates to a method of designing an ion channelblocker for an ion channel which includes providing an ion channelhaving an external vestibule portion and providing an antibody, bindingportion, probe, or ligand specific to the external vestibule portion ofthe ion channel, where the antibody, binding portion, probe, or ligandis effective to inhibit ion transport through the ion channel.

Another aspect of the present invention relates to a method ofinhibiting the ion transport through an ion channel having an externalvestibule portion which includes providing an ion channel blocker whichis specific to the external vestibule portion of the ion channel underconditions effective to inhibit ion transport through the ion channel.

Yet another aspect of the present invention relates to a method ofscreening a drug for effectiveness as an ion channel blocker for an ionchannel where the ion channel has an external vestibule portion. Themethod includes contacting a cell having an ion channel with an ionchannel blocker candidate and evaluating the cell to determine if theion channel blocker candidate binds to the external vestibule portion ofthe ion channel and inhibits ion transport through the ion channel.

Yet another aspect of the present invention relates to an antibody wherethe antibody, binding portion, probe, or ligand which inhibits iontransport of an ion channel by binding to an external vestibule portionof the ion channel.

A number of compounds useful in treating various diseases in animals,including humans, are thought to exert their beneficial effects bymodulating the functioning of ion channels. An understanding of thepharmacology of compounds that interact with ion channels, and theability to rationally design compounds that will interact with ionchannels to have desired therapeutic effects, have been hampered by thelack of rapid, effective means to identify those compounds whichinteract with specific ion channels. The availability of rapid,effective means to identify compounds which interact with ion channelswould enable the rapid screening of a large number of compounds toidentify those candidates suitable for further, in-depth studies oftherapeutic applications. With the method of the present invention,blockers for each specific ion channel based on available nucleotide oramino acid sequence information are rationally designed. Using aspecific antibody against the external vestibule of a channel protein,the specific blocking of this channel is achieved by this antibody, butother related channels are not blocked. This approach provides a methodto rationally design specific ion channel blockers.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show the S5-S6 linker region of Kv1.2, Kv1.3 and Kv3.1potassium channels. In FIG. 1A, the amino acid sequence alignment ofrKv1.2, hKv1.3 and rKv3.1 shows the S5 and S6 transmembrane domains, thepore-forming region (P region) and indicates the peptide used forgenerating the blocking antibody (underlined). Dashes represent sameamino acids as rKv1.2. In FIG. 1B, a schematic model is shown for theproposed arrangement of the external vestibule of Kv1.2. The peptideused for generating the blocking antibody is in bold and shaded. Onlythe flanking S5 and S6 domains and two of the four subunits of thechannel are illustrated. FIGS. 1C and 1D are a characterization ofKv1.2-BA and Kv1-NA antibodies. Membrane preparations from HEK-293cells, untransfected (lanes 1), transfected with Kv1.2 (lanes 2 and 4),or transfected with Kv1.3 (lane 3), were subjected to 10%SDS-polyacrylamide gel electrophoresis and were Western blotted withKv1.2BA antibody (FIG. 1C) or Kv1-NA antibody (FIG. 1D). Estimatedmolecular masses of Kv1.2 and Kv1.3 were 70 to 80 kDa. Bandsrepresenting endogenous Kv1 proteins could be seen upon longer exposure.FIG. 1E illustrates characterization of the Kv3.1-BA antibody. Membranepreparations from HEK-293 cells, untransfected (lane 1), transfectedwith Kv3.1 (lanes 2 and 4), or transfected with Kv1.2 (lane 3) weresubjected to SDS-PAGE and were Western blotted with Kv3.1-BA antibody.Lane 4 was probed with Kv3.1-BA that had been preincubated with thepeptide used to generate Kv3.1-BA. Estimated molecular mass of Kv3.1 was100 kD. Positions of prestained molecular mass markers in kilodaltonsare indicated on the left.

FIGS. 2A-2E show the blockage of Kv1.2 currents by Kv1.2-BA antibody.FIG. 2A shows Kv1.2 currents in Kv1.2 stably transfected HEK-293 cellsin the absence of Kv1.2-BA (HEK/Kv1.2 cells) or in the presence ofKv1.2-BA (+Kv1.2-BA). Untransfected HEK-293 cells have very smallendogenous currents (HEK). Addition of 250 nM Kv1.2-BA resulted in anabout 70% suppression of Kv1.2 currents. Whole-cell currents wereelicited by voltage pulses of 300-ms duration from a holding potentialof −70 mV. Current was measured in the range from −40 mV to 60 mVvarying the voltage in 10-mV steps. FIG. 2B shows a time course of theblockage of Kv1.2 currents by Kv1.2-BA (+Kv1.2-BA). In the absence ofKv1.2-BA, the Kv1.2 current is quite stable for over 15 minutes(HEK/Kv1.2). Peak currents (I) at +60 mV at each time point are comparedwith the currents (I_(o)) before the addition of the antibody. FIG. 2Cshows the current-voltage relationship of whole-cell currents. Allcurrents (I) are expressed as a fraction of the peak current (I_(max))at +60 mV in the absence of the antibody. FIG. 2D shows that Kv1.2-BAblockage is concentration-dependent. Data points were taken at 15minutes after the addition of antibody. The data are mean±standarddeviation of five to seven (n) experiments. FIG. 2E shows whole-cellpotassium currents from Kv1.2-transfected HEK-293 cells in the absenceof (HEK/Kv1.2) or in the presence (+Kv1.2-BA) of the blocking antibodyKv1.2-BA. Kv1.2-BA (250 nM) reduced the whole cell K+ currents by about70%. Preincubation of Kv1.2-BA with excess of the immunogenic peptide(Kv1.2-BA +peptide) reduced the suppression to the residual effect ofabout 25%. Preincubation of Kv1.2-BA with excess of a control peptide(the Kv1-NA immunogenic peptide) had no effects on Kv1-2-BA suppressionof Kv1.2 currents.

FIGS. 3A-3F illustrate a demonstration of the specificity of theblockage by Kv1.2-BA. FIG. 3A and 3B show that a control antibody,Kv1-NA (250nM), has a limited effect on Kv1.2 currents. FIGS. 3C and 3Dshow that Kv1.2-BA (250 nM) has limited effect on Kv1.3 currents inKv1.3 stably transfected HEK-293 cells. FIGS. 3E and 3F show thatKv1.2-BA (250nM) has no significant effect on Kv3.1 currents in Kv3.1stably transfected HEK-293 cells. The data are mean± standard deviationof five to seven experiments.

FIG. 4 illustrates the inhibition of ¹²⁵I-dendrotoxin binding byKv1.2-BA antibody. HEK-293 cells with stably transfected Kv1.2 channelswere incubated with ¹²⁵I-labeled α-dendrotoxin (10 nM; 290 Ci/mmol) inthe presence of the indicated concentrations of Kv1.2-BA antibody.Preimmune IgG did not have significant effects. At 500 nM, preimmune IgGproduced about 10% inhibition of ¹²⁵-dendrotoxin binding. Data areexpressed as fractions of dendrotoxin binding compared with controls inwhich no Kv1.2-BA antibody was added. Data are representative of threesimilar experiments.

FIGS. 5A and 5B show the blockage of endogenous Kv1.2 currents byKv1.2-BA in neuronal cells. FIG. 5A shows whole-cell potassium currentsfrom NG108-15 cells in the absence (NG108-15 cells) or presence(+Kv1.2-BA) of the blocking antibody Kv1.2-BA. Kv1.2-BA (250 nM) reducedthe whole-cell K+ currents by about 75%. FIG. 5B shows that the controlantibody Kv1-NA (250 nM) reduced the current by about 25%. Data arerepresentative of six similar experiments.

FIGS. 6A-6D shows the blockage of Kv3.1 currents by Kv3.1-BA antibody.FIG. 6A illustrates Kv3.1 currents in Kv3.1 stably transfected HEK-293cells in the absence (HEK/Kv3.1) or presence (+Kv3.1-BA) of Kv3.1-BA.Addition of 250 nM of Kv3.1-BA led to an about 80% suppression of Kv3.1currents. FIG. 6B illustrates that Kv3.1-BA blockage is concentrationdependent. Data points were taken at 15 minutes after the addition ofantibody. FIGS. 6C and 6D show that Kv3.1-BA has limited effects onKv1.2 currents in Kv1.2 stably transfected HEK-293 cells. The data aremean±SD of five to seven experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of designing an ion channelblocker for an ion channel which includes providing an ion channelhaving an external vestibule portion and providing an antibody, bindingportion, probe, or ligand specific to the external vestibule portion ofthe ion channel, where the antibody, binding portion, probe, or ligandinhibits ion transport through the ion channel.

Another aspect of the present invention relates to a method ofinhibiting the ion transport through an ion channel having an externalvestibule portion which includes providing an ion channel blocker whichis specific to the external vestibule portion of the ion channel underconditions effective to inhibit ion transport through the ion channel.

Yet another aspect of the present invention relates to a method ofscreening a drug for effectiveness as an ion channel blocker for an ionchannel where the ion channel has an external vestibule portion. Themethod includes contacting a cell having an ion channel with an ionchannel blocker candidate and evaluating the cell to determine if theion channel blocker candidate binds to the external vestibule portion ofthe ion channel and inhibits ion transport through the ion channel.

Yet another aspect of the present invention relates to an antibody wherethe antibody, binding portion, probe, or ligand which inhibits iontransport of an ion channel by binding to an external vestibule portionof the ion channel.

Ion channels are generally discussed in Jan, L. Y., et al., Annu. Rev.Neurosci., 20:91-123 (1997) and Jan, L. Y., et al., Annu. Rev. Physiol.,54:537-555 (1992), which are hereby incorporated by reference.

The ion channel blockers obtained in accordance of the present inventionare any compounds which will bind to the ion channel to inhibit iontransport through the ion channel. Preferably, the ion channel blockersbind to an external vestibule portion of the ion channel. The ionchannel blockers may be antibodies, binding portions thereof, probes, orligands. Typically, the ion channel blockers are antibodies.

Ion transport to be inhibited according to the present invention includeion transport through potassium, sodium, calcium, and chloride channels.Preferably, the portion of the ion channel to be blocked is the externalvestibule portion. Most preferably, the external vestibule portion islocated between the S5 transmembrane and the pore forming region of thechannel protein or between the pore forming region and the S6transmembrane of the channel protein (FIG. 1A). For example, theexternal vestibule portion is a 15 amino acid peptide from Kv1.2potassium channel having SEQ ID NO:1: Phe Ala Glu Ala Asp Glu Arg AspSer Gln Phe Pro Ser Ile Pro1               5                   10                  15

or a 14 amino acid peptide from Kv3.1 potassium channel having SEQ IDNO:3: Gly Ala Gln Pro Asn Asp Pro Ser Ala Ser Glu His Thr His1               5                   10

or a 15 amino acid peptide from Kv1.3 potassium channel having SEQ IDNO:4: Phe Ala Glu Ala Asp Asp Pro Thr Ser Gly Phe Ser Ser Ile Pro1               5                   10                  15or any other Kv related channels(Jan, L. Y. et al., Annu. Rev. Physiol.54:537-555(1992), which is hereby incorporated by reference).

The term “inhibit” or “inhibiting” as used in the present applicationrefers to the ability of the ion channel blocker of the presentinvention to prevent passage of (i.e. block) the ions through the ionchannel. Although not meaning to be bound be theory, it is believed thatthe channel blocker of the present invention binds to the target peptidesequence and physically blocks the permeation of ions through the pore.Alternatively, ion channel blocker binding could cause a conformationalchange in the channel protein that closes the pore.

Preferably, the channel is contained in a mammalian cell, such as aneuronal cell, a cardiac myocyte, a muscle cell, or any other excitablecell.

As indicated above, ion channel blockers which bind to the externalvestibule portion of an ion channel are utilized. These ion channelblockers include antibodies, such as monoclonal and polyclonalantibodies, antibody fragments, half-antibodies, hybrid derivatives,probes, or other molecular constructs.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The resulting fused cells, orhybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned, and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, Nature, 256:495 (1975), which is hereby incorporated byreference.

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the protein or polypeptide of the presentinvention. Such immunizations are repeated as necessary at intervals ofup to several weeks to obtain a sufficient titer of antibodies.Following the last antigen boost, the animals are sacrificed and spleencells removed.

Fusion with mammalian myeloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol.,6:511 (1976), which is hereby incorporated by reference.) This immortalcell line, which is preferably murine, but may also be derived fromcells of other mammalian species, including but not limited to rats andhumans, is selected to be deficient in enzymes necessary for theutilization of certain nutrients, to be capable of rapid growth, and tohave good fusion capability. Many such cell lines are known to thoseskilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 μl per site at sixdifferent sites. Each injected material will contain syntheticsurfactant adjuvant pluronic polyols, or pulverized acrylamide gelcontaining the protein or polypeptide after SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. Ultimately, the rabbits are euthenized with pentobarbital 150mg/Kg IV. This and other procedures for raising polyclonal antibodiesare disclosed in Harlow et. al., editors, Antibodies: A LaboratoryManual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, binding portions of suchantibodies can be used. Such binding portions include Fab fragments,F(ab′)₂ fragments, and Fv fragments. These antibody fragments can bemade by conventional procedures, such as proteolytic fragmentationprocedures, as described in Goding, Monoclonal Antibodies: Principlesand Practice, New York:Academic Press, pp. 98-118 (1983), which ishereby incorporated by reference.

The present invention also relates to probes found either in nature orprepared synthetically by recombinant DNA procedures or other biologicalprocedures. Suitable probes are molecules which bind to the externalvestibule portion of the ion channels identified by the antibodies ofthe present invention. Such probes can be, for example, proteins,peptides, lectins, or nucleic acid probes.

The external vestibule portions of the ion channels listed hereininclude sequences which are substantially the same as the sequenceslisted herein. Variations, may be made by, for example, the deletion oraddition of amino acids that have minimal influence on the properties,structure, or nature of the amino acid. Amino acid additions, deletions,and/or substitutions which do not negate the ability of the resultingprotein to raise an antibody capable of blocking an external vestibuleportion of an ion channel are within the scope of an amino acid sequencecorresponding to or having or as shown in a particular amino acidsequence. Such additions, deletions, and/or substitutions can be, forexample, the result of point mutations in the DNA encoding the aminoacid sequence, such point mutations made according to methods known tothose skilled in the art. Substitutions may be conservativesubstitutions of amino acids. As used herein, two amino acid residuesare conservative substitutions of one another where the two residues areof the same type. In this regard, for purposes of the present invention,proline, alanine, glycine, serine, and threonine, all of which areneutral, weakly hydrophobic residues, are of the same type. Glutamicacid and aspartic acid, which are acidic, hydrophilic residues, are ofthe same type. Another type of residue is the basic, hydrophilic aminoacid residues, which include histidine, lysine, and arginine. Leucine,isoleucine, valine, and methionine all of which are hydrophobic,aliphatic amino acid residues, form yet another type of residue. Yetanother type of residue consists of phenylalanine, tyrosine, andtryptophan, all of which are hydrophobic, aromatic residues. Furtherdescriptions of the concept of conservative substitutions are given byFrench et al., J. Molecular Evolution 19:171-175 (1983), Taylor, J.Theor. Biol. 119:205-218 (1986), and Bordo et al., J. Mol. Biol.217:721-729 (1991).

Any one of a number of methods well known in the art can be used toidentify a hybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, Western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell. Res. 175:109-124 (1988), which is hereby incorporated byreference).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass are determined using procedures known in the art.

Having described the ion channel blocker of the present invention, andmethods for designing the ion channel blocker, this ion channel blockercan be used in a method of screening a drug for effectiveness as an ionchannel blocker. The ion channel blocker which is specific to theexternal vestibule portion of the ion channel is contacted with a cellwhich contains ion channels. The cell is then evaluated to determinewhether the ion channel blocker inhibits ion transport through the ionchannel. From this evaluation, ion channel blockers which inhibit iontransport through the ion channel can be found. The ability of the ionchannel blocker to inhibit ion transport through the ion channel can beassayed according to methods known in the art, such as voltage clampanalysis of the channel as described in Huang et al., Cell 75:1145-1150(1993), which is hereby incorporated by reference.

Thus, the present invention may screen drugs useful in treating diseasedstates, such as hypertension, cardiac ischemia, cerebral ischemia,bronchi construction, and neurological diseases.

EXAMPLES

Materials and Methods

Affinity-Purified Polyclonal Antibodies and Ommunoblot.

Kv1.2-BA, Kv1-NA, and Kv3.1-BA rabbit polyclonal antibodies were madeand affinity purified through Genemed Biotechnologies, Inc. (South SanFrancisco, Calif.). A cysteine residue was added to the carboxyl end ofthe peptide having SEQ ID NO:1: Phe Ala Glu Ala Asp Glu Arg Asp Ser GlnPhe Pro Ser Ile Pro1               5                   10                  15

or the amino end of the peptide having SEQ ID NO:2: Asp Pro Leu Arg AsnGlu Tyr Phe Phe Asp Arg Asn Arg Pro Ser1               5                   10                  15

or the carboxyl end of the peptide having SEQ ID NO:3: Gly Ala Gln ProAsn Asp Pro Ser Ala Ser Glu His Thr His1               5                   10for keyhold limpet hemocyanin conjugation. Rabbit antiserum was purifiedwith the peptide-affinity matrix. The specificity of these antibodieswas confirmed by competition experiments with different peptides duringimmunoblotting. Immunoblot of membrane proteins (30 μg per lane) fromHEK and transfected HEK cells was done as described, with somemodifications (Langhans-Rajasekaran, S. A. et al., Proc. Natl. Acad.Sci. USA 92:8601-8605 (1995); Wan Y. et al., Nature, 380:541-544 (1996),which are hereby incorporated by reference). Boiling lysis solution (1%Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA,0.5% NP-40, 0.1% SDS, 1% Na-deoxycholate) was used to resuspend membraneproteins.

Electrophysiological Recordings with HEK-293 Cells.

Maintenance, growth, and stable transfection of HEK-293 cells wereperformed as described (Huang, X. Y. et al., Cell 75:1145-1156 (1993);Langhans-Rajasekaran, S. A. et al., Proc. Natl. Acad. Sci. USA92:8601-8605 (1995), which are hereby incorporated by reference). Ateach passage, some cells were plated onto 2.5-cm culture dishes withsmall cover slips for electrophysiological measurements.Electrophysiological recordings were made on the same day. Whole-cellpatch-clamp recordings were performed as described, with somemodifications (Huang, X. Y. et al., Cell 75:1145-1156 (1993), which ishereby incorporated by reference). Recordings were done at roomtemperature with pipettes pulled from micro-hematocrit capillary tubeswith resistances of 2 to 4 MΩ. The pipette solution contained 180 mMK-Asp, 5 mM NaCl, 5 mM Na-HEPES, 5 mM EGTA, 0.28 mM CaCl₂, 0.8 mM MgCl₂,1.5 mM ATP, 0.1 mM GTP, pH adjusted to 6.7 with HCl. The bath solutioncontained 118 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mMNa-HEPES, 23 mM Glucose, 54 mM Sucrose, pH adjusted to 7.4 with NaOH.Whole-cell currents were elicited by voltage pulses of 300-ms durationfrom a holding potential of −70 mV. Current was measured in the rangefrom −40 mV to 60 mV, varying the voltage in 10-mV steps. Seriesresistance values after seal formation were less than 3 MΩ and wereelectronically compensated. The current signals were low pass filteredat 5 kHz and leak subtracted. The data were collected by using Axopatch200A via pCLAMP6 (Axon, Forster City, Calif.) To investigate the effectsof antibodies on membrane potassium currents, cells were patched for 5minutes to obtain a stable baseline before antibodies were added to therecording chamber.

Electrophysiological Recordings with Neuronal Cells.

Maintenance and growth of the neuronal NG108-15 cells are as described(Huang, X. Y. et al., Cell 75:1145-1156 (1993); Han, H. Q. et al.,Nature, 349:697-700 (1991), which are hereby incorporated by reference).At each passage, some cells were plated onto 2.5-cm collagen-coatedculture dishes with small cover slips for electrophysiologicalmeasurements. Electrophysiological recordings were made about 3 to 7days after differentiation. Whole-cell patch-clamp recordings ofpotassium currents were performed as described (Wilk-Blaszczak, M. A. etal., Neuron, 13:1215-1224 (1994), which is hereby incorporated byreference). Recordings were done at room temperature using a 2 to 4 MΩresistance electrode. The pipette solution contained 115 mM KCl, 0.1 mMMgCl₂, 40 mM HEPES, 3 mM ATP, 0.1 mM GTP, pH adjusted to 7.3 with KOH.The bath solution contained 125 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mMMgCl₂, 20 mM HEPES, 5 mM Glucose, pH adjusted to 7.4 with NaOH.Recording protocol, data collection, and analysis were done as describedabove for HEK-293 cells.

Dendrotoxin Binding Assay.

Binding assay was performed as previously described for agonist bindingto muscarinic receptors (Wan et al., J. Biol. Chem. 272:17209-17215(1997), Wan et al., Nature 380:541-544 (1996); and Bence et al., Nature389:296-299 (1997), which are hereby incorporated by reference).Briefly, HEK293 cells stably expressing Kv1.2 channel proteins weregrown in 12-well dishes. After being washed two times with Hank'sbalanced salt solution (“HBSS”) plus 10 mM HEPES (pH 7.4), 300 μlbinding solution was added to each well. The binding solution containsHBSS plus 10 mM HEPES (pH 7.4), 1 mg/ml BSA, 1 mg/ml bacitracin, 1 mMPMSF, 10 nM ¹²⁵-α-dendrotoxin (290 Ci/mmol) (Amersliam Corp., ArlingtonHeights, Ill.; or Alomone Labs, Jerusalem, Israel), and variousconcentrations of Kv1.2-BA antibody. After incubation for 4 hours at 4°C., cells were washed two times with HBSS plus 10 mM HEPES (pH7.4) toremove unbound ¹²⁵-dendrotoxin. Cells were then lysed with 1 ml of 0.4 NNaOH and transferred to borosilicate tubes. The bound ¹²⁵-dendrotoxinwas counted with a gamma counter. Nonspecific binding of ¹²⁵-dendrotoxinto cells was determined in the presence of excess (10 μM) unlabeleddendrotoxin. Nonspecific inhibition of antibody on dendrotoxin bindingwas examined in the presence of 500 nM of preimmune IgG with<10%inhibition.

Data Analysis

Data for multisite inhibition can be expressed in terms of a Hillequation (Segel, I. H. Enzyme Kinetics, John Wiley & Sons, Inc., NewYork, (1993), which is hereby incorporated by reference):log {I/I _(o)/(1-I/I _(o) }=−n log[antibody]+log K′where I_(o) is the current amplitude before the addition of theantibody; I is that after the addition of the antibody; [antibody] isthe concentration of the antibody; and n indicates the number of bindingsites of the antibody on the channel. IC₅₀ is related to K′ as IC₅₀n=K′. A Hill plot for the inhibition by both Kv 1.2-BA or Kv3.1-BAantibodies gave the slope of the Hill plot −0.75, indicating only oneantibody binding to one channel complex. Thus, the Hill equation can besimplified as:I/I _(o)=1/(1+[antibody]/K′).All curve fittings were performed using this equation.

Example 1 Suppression of Kv1.2 Current by Kv1.2-BA Antibody

To test whether antibodies specific for peptides around the pore-regionsof individual ion channels can be used as a channel blocker, theblocking ability of an affinity-purified polyclonal antibody generatedagainst a 15 amino-acid peptide of a delayed-rectifier potassium channelKv1.2 (Huang, X. Y. et al., Cell 75:1145-1156 (1993)) (FIG. 1A) wasexamined. This sequence identified as SEQ ID NO:1: Phe Ala Glu Ala AspGlu Arg Asp Ser Gln Phe Pro Ser Ile Pro1               5                   10                  15located between the S5 transmembrane and the pore-forming (P) region, isvery likely part of the external vestibule (or the outer mouth) of thechannel protein (FIG. 1B) (Lu, Q. et al., Science 268:304-307 (1995);Hidalgo, P. et al., Science 268:307-310 (1995); Aiyar, J. et al., Neuron15:1169-1181 (1995), which are hereby incorporated by reference). Kv1.2was stably expressed in a mammalian cell line, HEK-293 cells (Huang, X.Y. et al., Cell 75:1145-1156 (1993), which is hereby incorporated byreference). The affinity-purified polyclonal antibody (Kv1.2-BA)detected Kv 1.2 protein in plasma membranes of Kv1.2-transfected cells(FIG. 1C). Whole-cell patch-clamp recording revealed large outwardpotassium currents, elicited at depolarizing voltages, that are absentin untransfected HEK-293 cells (FIG. 2A). Addition of 250 nM of theaffinity-purified polyclonal antibody (Kv1.2-BA) to the externalsolution blocked about 70% of the Kv1.2 currents (FIG. 2). The currentamplitude decline, seen only after addition of Kv1.2-BA, occurred within1 minuteand reached about 70% suppression in about 7-15 minutes (FIG.2B). The Kv1.2 current is stable in the absence of antibodies (FIG. 2B).The current is decreased by the antibody Kv1.2-BA for all voltages≧0 mV(FIG. 2C). Also, the suppression was antibody concentration-dependent:with a rise in concentration of Kv1.2-BA, the inhibition of Kv1.2currents increases (FIG. 2D). The data for the channel inhibition by theblocking antibody could be well fitted by an equation for 1:1 bindingbetween the antibody and the Kv1.2 channel with an IC₅₀ of 54 nM (seedata analysis in Materials and Methods). This IC₅₀ is similar to theK_(d) of most potassium channel neurotoxins. Thus, the antibody Kv1.2-BAcan suppress the Kv1.2 current in Kv1.2 transfected cells.

Example 2

To ensure that the Kv1.2-BA antibody does indeed bind to the externalregion of Kv1.2 channel protein and that the blocking effect is due tothe binding of the Kv1.2-BA antibody to the channel protein, Kv1.2-BAantibody was preincubated with the immunogenic peptide that was used togenerate Kv1.2-BA. If blocking is due to binding of the antibody to thepeptide sequence in the external vestibule of the channel protein,preincubation with the peptide should prevent the inhibition. As shownin FIG. 2E, addition of 250 nM Kv1.2-BA after preincubation with theimmunogenic peptide only produced about 25% inhibition. Preincubationwith a control peptide did not inhibit the Kv1.2-BA-induced suppressionof Kv 1.2 currents (FIG. 2E). Thus, the majority of the inhibition byKv1.2-BA on Kv1.2 currents is due to specific finding of Kv1.2 to theparticular peptide sequence around the pore region of Kv1.2 channels.The residual about 25% inhibition is likely due to either nonspecificblocking of the channel by high concentrations of proteins in the bathsolution or the presence of another antiserum in the polyclonal antibodypreparation that fortuitously recognize sequences of the channelprotein. Nonetheless, Kv 1.2-BA can significantly block Kv1.2 currentsin Kv1.2-transfected cells.

Example 3

No Effect of Kv1.2 Current by a Control Antibody Kv1-NA

To further exclude the possibility that the Kv1.2 BA blocking effectswere non-specific and that any antibody added outside cells somehowinterferes the channel function, the effects on the Kv1.2 currents byanother affinity-purified polyclonal antibody (Kv1-NA) was examined(FIG. 3A and 3B). Kv1-NA was generated against a peptide having SEQ IDNO:2: Asp Pro Leu Arg Asn Glu Tyr Phe Phe Asp Arg Asn Arg Pro Ser1               5                   10                  15from the intracellular N-terminus of Kv1.2, which is identical in allmembers of the Kv1 family (FIG. 1D). Addition of this control antibodyKv1-NA had no effect at low concentrations (<20 nM) and very smalleffects at very high concentrations (>100 nM) on the Kv1.2 currents(FIG. 3B). These results indicate that the effect of antibody Kv1.2-BAis specific.

Example 4 Effects of Related Kv1.3 or Kv3.1 Currents by Kv1.2-BAAntibody

To additionally ensure the specificity, the Kv1.2-BA antibody was testedon related potassium channels. Kv1.3 belongs to the same subfamily ofdelayed-rectifier potassium channels as Kv1.2. Kv1.3 has the samesequence around the pore-region as Kv1.2, with the exception of fiveamino acids (FIG. 1A). When stably expressed in HEK-293 cells, Kv1.3exhibits C-type slow inactivation (Attali, B. J. Biol. Chem.267:8650-8657 (1992); Panyi, G. et al. Biophys. J. 68:896-903 (1995),which are hereby incorporated reference) (FIG. 3C). In contrast to theresults obtained with Kv1.2 currents, addition of Kv1.2-BA had littleeffects on Kv1.3 currents, only small effect at high concentrations(similar to the residual effect of Kv1.2 currents by Kv1.2-BApreincubated with the immunogenic peptide) (FIG. 3C and 3D). Kv3.1 isanother delayed-rectifier K⁺ channel, but belongs to a differentsubfamily (Yokoyama, S. et al. FEBS Letters 259:37-42 (1989); Luneau, C.J. et al. Proc. Natl. Acad. Sci. USA 88:3932-3936 (1991), which arehereby incorporated by reference). Although Kv3.1 has high sequencehomology with Kv1.2 in transmembrane domains and pore-forming regions,it differs from Kv1.2 in the region where Kv1.2-BA binds (FIG. 1A). Aswould be expected, there was only a small change of Kv3.1 currentsexpressed in HEK-293 cells after addition of Kv1.2-BA (FIGS. 3E and 3F).These experiments demonstrated that Kv1.2-BA specifically blocks theKv1.2 currents, without affecting other related K⁺ currents.

Example 5 Inhibition of Binding of ¹²⁵I-Dendrotoxin by Kv1.2-BA

Kv1.2 channel protein is one of the major receptors for dendrotoxin invivo (Scott, J. Biol. Chem. 265:20094-20097 (1990)), which is herebyincorporated by reference). Since α-dendrotoxin also binds to theexternal vestibule of Kv1.2 channel protein (Hurst, R. S. Mol.Pharmacol. 40:572-576 (1991), which is hereby incorporated byreference), it is expected that binding by α-dendrotoxin and Kv1.2-BAantibody should be mutually exclusive. If the Kv1.2 channel protein isfirst bound with the Kv1.2-BA antibody, then binding of α-dendrotoxin toKv1.2-BA channel protein should be decreased compared to the binding inthe absence of Kv1.2-BA antibody due to the occupancy of the channelproteins by the antibody that excludes the binding of α-dendrotoxin. Asshown in FIG. 4, increased concentrations of Kv1.2-BA led to theinhibition of ¹²⁵I-α-dendrotoxin binding in Kv1.2-transfected HEK cells.Thus, this data further demonstrates that Kv1.2-BA blocks Kv1.2 currentsby binding to the external vestibule of Kv1.2 channel protein.

Example 6 Suppression of Endogenous Kv1.2 Current by Kv1.2-BA Antibody

The method of the present invention was tested in a neuronal cell line.Previously, Kv1.2 had also been cloned by screening a cDNA library madefrom the neuronal NG108-15 cells, and it has been shown that Kv1.2 isabundantly expressed in NG108-15 cells (Yokoyama, S. et al. FEBS Letters259:37-42 (1989), which is hereby incorporated by reference). Therefore,whole-cell patch-clamp analysis was performed on NG108-15 cells (Han.H.Q. et al. Nature 349:697-700 (1991); Wilk-Blaszcak, M. A. et al.,Neuron 13:1215-1224 (1994), which are hereby incorporated by reference)(FIG. 5). Addition of the blocking antibody Kv1.2-BA (250 nM) resultedin a 76±12% (n=6) decrease of the whole-cell potassium currents within10-15 minutes, a significantly (P<0.002, paired t test) higherinhibition than that caused by the control antibody Kv1-NA (25±8%[n=6])decrease over the same period with 250 nM Kv1-NA). Untreated cellsshowed a 8±3%(n=6) decrease. These results demonstrate that the Kv1.2-BAantibody blocks endogenous Kv1.2 current as well as heterologouslyexpressed Kv1.2 currents.

Example 7 Application of the Method to Another Subfamily of K⁺ Channels

To extend the method of the present invention to other subfamilies of K⁺channels, another polyclonal antibody was generated against a 14amino-acid peptide, having SEQ ID NO:3: Gly Ala Gln Pro Asn Asp Pro SerAla Ser Glu His Thr His 1               5                   10from the external vestibule of the Kv3.1 channel protein (FIG. 1A and1E). This affinity-purified anti-Kv3.1 antibody (Kv3.1-BA) detectedKv3.1 protein in plasma membranes of Kv3.1-transfected HEK-293 cells(FIG. 1E). Kv3.1-BA at 250 nM blocked about 79% of the Kv3.1 currentsfrom Kv3.1-transfected HEK cells (FIG. 6A). The Kv3.1 current is stablein the absence of antibodies. The suppression is antibody dose dependent(FIG. 6B). The IC₅₀ is 58 nM. This Kv3.1-BA antibody had limited effectson Kv1.2 currents (FIGS. 6C and 6D). Thus, the antibody Kv3.1-BA canspecifically suppress Kv3.1 currents.

Discussion

In practice, like any other methods using antibodies, the concentrationused is important and controls are essential. It is useful to testseveral concentrations of antibodies to obtain appropriate amounts tomaximize inhibition and minimize nonspecific background suppression. Ifthe antibody is used at too high a concentration, high background(nonspecific suppression) can result. If the antibody concentration istoo low, the inhibition will be too small to be certain of specificity.In general, the optimal concentrations for purified polyclonalantibodies will be in the range of about 20-60 nM. At concentrations<60nM, the specific antibodies could reduce the currents significantly (ashigh as 50%), while nonspecific antibodies produced no suppression otherthan rundown (<10%). When examining endogenous currents, it is veryimportant to compare the suppression produced by the channel-blockingantibody with a control antibody, or one can perform the experiments inthe presence and absence of the immunogenic peptide used to generate theantibody and compare the differences.

Given the remarkable specificity and selectivity of antibodies for theirtargets, the method of the present invention provides a superiorapproach to rationally design specific ion channel blockers. A largenumber of K⁺ channels expressing similar properties makes it a difficulttask to identify a native K⁺ current as one expressed by a cloned K⁺channel gene. At present time, the identification of a particularcurrent in cells possessing a broad array of overlapping currentsdepends on a limited array of pharmacological tools. Although theavailability of mutants is of great help for such an endeavor, there arenot many K⁺ channel mutants identified in vertebrates. Approachestargeted at the biosynthesis or assembly of channel proteins such asintroducing antisense oligonucleotides or expressing dominant negativemutant channels in native cells have limitations, for example, thedependence on the turnover rate of endogenous channel proteins (Tu, L.et al. (1995) Biophys. J. 68:147-156; Chung, S. et al. (1995) Proc.Natl. Acad. Sci USA 92:5955-5959). Since the method of the presentinvention is aimed at the functional membrane channel proteins, itavoids such limitation. Furthermore, antibodies have been raised thatdetect single amino acid substitutions, or differentiate betweenphosphorylated and unphosphorylated peptides (Harlow, E. Antibodies(Cold Spring Harbor Laboratory, 1988). Thus, they are excellent tools todiscriminate among structurally related ion channels.

The biophysical mechanism of the blocking ability of Kv1.2-BA on Kv1.2currents or Kv3.1-BA on Kv3.1 currents is not yet clear. The targetpeptide sequence is close to the ion flux pathway, and it is likely thatthe antibody binds to the peptide and physically blocks the permeationof K⁺ ions as is the case with sorption toxins (MacKinnon, R. et al., J.Gen. Physiol. 91:335-349 (1988), which is hereby incorporated byreference). Alternatively, antibody binding could cause a conformationalchange in the channel protein that closes the pore. Regardless of themechanism, however, the present invention is a novel approach toidentify a specific ion channel involved in producing a particularendogenous current. This immuno-electrophysiological approach shouldalso be applicable to other ion channels. Also, this method can be usedor modified as a rational drug design using ion channels as therapeutictargets. In addition, it should be possible to design channel openers bygenerating antibodies that force the channel into an open configuration.Furthermore, using the same peptide or other nearby peptides as probes,it is possible to screen for small peptides or oligonucleotides thatcould be used as ion channel blockers using combinatorial chemistry.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An antibody, binding portion, probe, or ligand which inhibits iontransport of an ion channel by binding to an external vestibule portionof the ion channel.
 2. The antibody, binding portion, probe, or ligandaccording to claim 1, wherein the ion channel is a potassium channel, asodium channel, a calcium channel, or a chloride channel.
 3. Theantibody, binding portion, probe, or ligand according to claim 2,wherein the ion channel is a mammalian ion channel.
 4. The antibody,binding portion, probe, or ligand according to claim 3, wherein the ionchannel is from an excitable cell.
 5. The antibody, binding portion,probe, or ligand according to claim 2, wherein the ion channel is a Kvion channel.
 6. The antibody, binding portion, probe, or ligandaccording to claim 5, wherein the antibody is a polyclonal antibody. 7.The antibody, binding portion, probe, or ligand according to claim 6,wherein the ion channel is a Kv1.2, Kv1.3, or Kv3.1 ion channel.
 8. Theantibody according to claim 7, wherein the external vestibule portionhas a sequence corresponding to SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:4.